Revolutionary advances in biotechnology and genetic engineering have created enormous potential for marketing cellular by-products, including for example, proteins, including protein pharmaceuticals such as interferon, monoclonal antibodies, TPA (Tissue Plasminogen Activator), growth factors, insulin, and cells for transplantation. The demand for these products has grown tremendously and will continue to do so, creating a need for efficient methods of producing industrial quantities of cell-derived pharmaceuticals and other products. Further, the demand for efficient methods of analyzing and isolating biological products through chromatographic technology, and the need to improve bio-implantables continues to grow.
Research and study of cell structure and morphology are fundamental to continued progress in the diagnosis and treatment of human diseases. Numerous cell products are of vital importance therapeutically and commercially, including, for example, hormones, enzymes, viral products, vaccines, and nucleic acids. The production of these products requires large scale cell culture systems for their production.
Mammalian cells can be grown and maintained in vitro, but are generally anchorage-dependent, i.e., they require a solid surface or substrate for growth. The solid substrate is covered by or immersed in a nutrient medium particular to the cell type to be cultured. The nutrient medium and solid substrates are contained in a vessel and provided with an adequate supply of oxygen and carbon dioxide to support cell growth and maintenance. Cell cultures may be batch systems, in which nutrients are not replenished during cultivation although oxygen is added as required; fed batch systems, in which nutrient and oxygen are monitored and replenished as necessary; and perfusion systems, in which nutrient and waste products are monitored and controlled (Lubiniecki, Large Scale Mammalian Cell Culture Technology, Marcel Dekker, Inc., New York, 1990).
The primary commercial systems used for mammalian cell culture use solid matrix perfusion and microcarrier bead systems (Lubineicke, supra). The solid matrix perfusion systems utilize glass columns packed with glass beads or helices, which form a matrix as the solid substrate for cell growth. Once cells have attached to the matrix, medium is continuously recycled from a storage vessel for support of cell growth and maintenance. A similar perfusion system uses hollow fibers as the solid matrix instead of beads.
In microcarrier systems, small spheres are fabricated, for example, from an ion exchange gel, dextran, polystyrene, polyacrylamide, or collagen-based material. These materials have been selected for compatibility with cells, durability to agitation and specific gravities that will maintain suspension of the microcarriers in growth mediums. Microcarriers are generally kept in suspension in a growth medium by gently stirring them in a vessel. Microcarrier systems are currently regarded as the most suitable systems for large-scale cell culture because they have the highest surface to volume ratio and enable better monitoring and control. Nevertheless, current microcarrier culture systems have a number of serious disadvantages: small microcarrier cultures cannot be used to inoculate larger microcarrier cultures; therefore, a production facility must use other culture systems for this purpose; the cost of microcarriers is high, which can necessitate reprocessing of the microcarriers for reuse with the attendant costs; and the oxygen transfer characteristics of existing microcarrier systems are rather poor.
Specific forms of calcium phosphate ceramic have been identified for use in microcarriers to support anchorage-dependent cells in suspension. These specialized ceramics provide a material, which is biomimetic, i.e., it is composed of mineral species found in mammalian tissues, and which can be further applied to a variety of in vitro biological applications of commercial interest. A number of common cell lines used in industrial applications require attachment in order to propagate and need substrate materials such as microcarriers for large scale cultivation. U.S. Pat. No. 4,757,017 (Herman Cheung) describes the use of solid substrates of mitogenic calcium compounds, such as hydroxylapatite (HA) and tricalcium phosphate (TCP) for use in in vitro cell culture systems for anchorage-dependent mammalian cells. The unique features of this technology include the growth of cells in layers many cells thick, growth of cells that maintain their phenotype and the ability to culture cells for extended periods of time. Cheung demonstrated the application of this technology for culturing red blood cells. A current limitation of this technology is that the microcarriers are only available in a non-suspendable granular form. The density of these microcarriers further limits the ability to scale-up this technology for large bioreactors, which require a suspendable microbead carrier. Cheung, also describes the use of large substrates in monolithic forms for the culture of cells, but he does not identify methods for producing large area monoliths conforming to the contours and sizes of tissues to be replaced in vivo or grown in vitro . A complementary system using an aragonite (CaCO3) is described in U.S. Pat. No. 5,480,827 (G. Guillemin et. al). Although this patent also mentions the importance of calcium in a support system for mammalian cell culture, the calcium compound was not in a suspendable form. Likewise, Guillemin et. al. do not identify methods for producing large area monoliths conforming to the contours and sizes of tissues to be replaced in vivo or grown in vitro.
The concept of fabricating a suspendable microcarrier bead with a minor component of glass was discussed by A. Lubiniecki in Large-Scale Mammalian Cell Culture Technology in which a minimal glass coating was applied to a polymer bead substrate by a chemical vapor deposition process or low temperature process. This approach also was disclosed in U.S. Pat. No. 4,448,884 by T. Henderson (see also U.S. Pat. Nos. 4,564,532 and 4,661,407). However, this approach primarily used the polymer bead substrate to maintain suspendability.
An example of the use of non-suspendable or porous ceramic particles for cell culture is taught by U.S. Pat. No. 5,262,320 (G. Stephanopoulos) which describes a packed bed of ceramic particles around and through which oxygen and growth media are circulated to encourage growth of cells. U.S. Pat. No. 4,987,068 (W. Trosch et. al.) also teaches the use of porous inorganic (glass) spheres in fixed bed or fluidized bed bioreactors. The pores of the particles act as sites for the culture of cells. Conversely, Richard Peindhl, in U.S. Pat. No. 5,538,887, describes a smooth surface cell culture apparatus which would limit cell attachment to chemical adhesion and prevent mechanical interlocking.
Macroporous glass beads also have been reported by D. Looby and J. Griffiths, “Immobilization of Cells In Porous Carrier Culture”, Trends in Biotechnology, 8:204209, 1990, and magnesium aluminate porous systems by Park and Stephanopolous, “Packed Bed Reactor With Porous Ceramic Beads for Animal Cell Culture”, Biotechnology Bioengineering, 41: 25-34,1993. Fused alumina foams have been reported by Lee et. al., “High Intensity Growth of Adherent Cells On a Porous Ceramic Matrix.” Production of biologicals from Animal Cells in Culture, editors, R. E. Butterworth-Heinemann et. al., pp. 400-405, 1991, and polyurethane foam by Matsushita et. al., “High Density Culture of Anchorage Dependent Animal Cells by Polyurethane Foam Packed Bed Culture Systems”, Applied Microbiology Biotechnology, 35:159-64, 1991.
Fluidized bed reactors have been used for cell culture as reported by J. M. Davis (editor), Basic Cell Culture, (Cartwright and Shah), Oxford University Press, New York, 1994, but require carrier systems with densities between 1.3 and 1.6 g/cc. According to Cartwright (J. M. Davis, supra.), generally, in fluidized beds, cells do not grow on the exterior surface of carriers where they would be dislodged by interparticle abrasion. Instead, as with macroporous microcarriers, they colonize the interior pores where they proliferate in a protected microenvironment. As examples, (Cartwright, supra, p. 78) cell carriers used in fluidized beds include glass beads (Siran by Schott Glass), and collagen microspheres produced by Verax. Cartwright also describes other conventional microcarriers weighted with TiO2 (Percell Biolytica products) and LAM-carrier polyethylene beads weighted with silica.
Hydroxylapatite and calcium phosphates have been used for implant applications with and without bone mixtures and bone growth factors. For example, Jarcho, Dent. Clin. North Am. 30:25-47 (1986) describes implanting of calcium phosphates to augment bone, while Ripamonti et. al., MRS Bulletin 36-39 (November, 1996) describes augmentation of bone with bone morphogenic proteins (BMP's), including TGF-beta, BMP's 108, OP-1 and 2, and dimineralized bone matrix with and without hydroxylapatite. Other growth factors applicable for bone augmentation are also set forth by Lane et. al., Clinical Orthopaedics and Related Research., 367S, S107-117 (October 1999), and include recombinant human bone morphogenetic protein (rhBMP), fibroblast growth factor (FGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), and growth differentiation growth factor (GDF). Various types of calcium implants, including tricalcium phosphate, hydroxylapatite, calcium phosphate and calcium carbonate, are listed in Han et. al., J. Western Soc. Periodontology:Perio Abstracts 32(3):88-108 (1984). The use of dense and porous hydroxylapatite with and without grafted bone is reported in Matukas et. al., J. Neurosurgery, 69:514-517 (1988). Similarly, Small et. al., Intl J. of Oral Maxillofacial Implants, 8(5):523-528 (1993) describes hydroxylapatite particles used with freeze dried bone to augment bone for implants. Also, Hollinger, United States Army Institute of Dental Research Information Bulletin, 4(2) (Winter, 1990), lists grafting materials that include various commercial types of hydroxylapatite, tricalcium phosphates and bone grafts. Finally, several investigators have demonstrated the culturing of marrow tissues, stem cells, and periosteal-derived cells on calcium phosphate materials, which have subsequently been implanted in animal models to produce the formation of bone and/or cartilage tissue by osteochondrogenic induction. These investigators include Nakahara et. al., Clinical Orthopaedics, (276):291-8 (Mar. 1992); Grundel et. al., Clinical Orthopaedics, (266):244-58 (May 1991); Toquet et. al., Journal of Biomedical Research, 44(1):98-108 (Jan. 1999); Bruder et. al., Journal of Bone and Joint Surgery, 80(7):985-96 (July 1998). However, none of these publications describe the use of hollow calcium-containing microspheres as implantable substrates. All of these approaches to the culture of anchorage dependent cells suffer from either the inability to grow cells in a suspendable environment or to allow an encapsulated volume in which cells can be grown or have a proven viablility as an implant material. Furthermore, the mass of material implanted as a carrier of cellular components is generally in excess of what can be resorbed in a ready manner.
Bonding agents applied as coatings can be used in conjunction with hollow microspheres are sited in WO98/43558, and include both polymeric and calcium based cements. Resorbable polymers which are applicable for orthopedic augmentation applications have been summarized by Behravesh et. al., Clinical Orthopaedics and Related Research, 367S, S118-125 (October 1999), and include polylactic acid, polygalatic acid, polycaprolactone, poly α-hydroxy esters, polyphosphazenes, polyanhydrides, and polypropylene fumarate. U.S. Pat. No. 5,522,893 (Chow et. al), sets forth a review of methods for making calcium phosphate cements, which can be employed for bone augmentation applications. Likewise, U.S. Pat. Nos. 4,612,053 (Brown et. al) and 5,047,031 (Constantz et. al) establish alternative and complementary methods of calcium phosphate cement formulation. Wright Medical Technology, Inc.(Memphis, Tenn.) has also produced a granular calcium phosphate for bone augmentation called Osteoset® and calcium sulfate used as a cement to incorporate a demineralized bone matrix called Allomatrix™ and further cites in its literature that calcium sulfate has been used for over 100 years as a bone void filler. Generally, cements used for bone augmentation applications lack sufficient porosity to allow for optimal bone in-growth and release of biological agents or live cells.
An excellent review of nitric oxide in biological applications is cited in the Encyclopedia of Inorganic Chemistry, Volume 5, 2482-2498, John Wiley & Sons (1994) and Nitric Oxide in Health and Disease, by J. Lincoln, Cambridge University Press, Cambridge, N.Y. & Melbourne, 1997. These references cite that nitric oxide (NO) is naturally produced in the body and is used on the cellular level as a defense against invading organisms, as a regulator of vascular tone and as a neuronal signaling agent. The concentration of nitric oxide and the presence of biological inhibiting agents or promoting agents determine the ultimate outcome, whether promoting tissue proliferation, health or tissue destruction.
According to these references, there are three types of cellular precursors to NO. They are all identified as nitric oxide synthases (NOS): Type I is associated with neurones, Type II is associated with a variety of cell types, but is primarily associated with host response to infection or invading organisms. Type III is primarily associated with endothelial cells.
NO has been implicated in increasing blood flow by arterial dilation, which can beneficially impact heart function, penile erection, and maintenance of blood supply to peripheral organs (Ziche et. al., Journal of Clinical Investigation, 94, 2036-44. According to J. Lincoln, Nitric Oxide in Health and Disease, Cambridge University Press, Cambridge, N.Y. & Melbourne, 1997, NO and NO antagonist have been used to treat asthmatic conditions. Likewise, NO has been used to increase nerve function (Schuman et. al., Annual Reviews of Neuroscience, 17, 153-83, 1994). Conversely, in larger concentrations, NO has been documented to be cytotoxic to tumor formations (Rocha et. al., International Journal of Cancer, 63, 405-11, 1995). NO has also been suggested to play a number of roles in renal function including renal blood flow, renin secretion and pressure induced natriuresis and diaresis (Bachmann et. al., American Journal of Kidney Diseases, 24, 112-29, 1994). NO has also been implicated as a growth factor (J. Lincoln, Nitric Oxide in Health and Disease, Cambridge University Press, Cambridge, N.Y. & Melbourne, 1997). Nitric oxide can also be used to treat pathogens and other invading organisms as set forth by James, S. L., Microbiological Reviews, 59, 533-47, 1995.
Tam et. al., Life Sciences, 51, 1277-84, 1992 describes a method of producing NO from sodium nitrite and hydrochloric acid. NO can also be purchased as a purified gas from commercial suppliers such as Matheson Gas Products, Cucamonga, Calif. Also, Manahan in Environmental Chemistry, 6th Edition, Lewis Publishers (1994) describes methods for the formation of NO at high temperatures from N2 and 02 in controlled atmospheres. Ishii et. al., American Journal of Physiology, 261, 598-603, 1991 describes a method of producing NO from sodium nitrite and hydrochloric acid.
The treatment of biological disorders with polymeric compounds binding NO compositions is set forth in U.S. Pat. No. 5,718,892 (Keefer et. al). This patent further demonstrates the usefulness of delivering NO for other medical applications. Other precursor forms of NO compounds include nitric oxide synthase or L-arginine. Also, superoxide dismutase causes an increase in the production of free NO as cited by Hobbs et. al., Proceedings of the National Academy of Sciences USA, Nov. 8, 1991, (23): 10992-6, 1994. Furthermore, NO is a physiological messenger and cytotoxic agent dependent on other mediating agents (J. Lincoln, Nitric Oxide in Health and Disease, Cambridge University Press, Cambridge, N.Y. & Melbourne, 1997).
NOS (nitric oxide synthases) Types I and III depend on elevated Ca2+ levels to become activated, and Type II is regulated to a lesser degree by the presence of Ca2+ (Jordan et. al., Surgery, 118, 138-45, 1995). These references together indicate that all three types of nitric oxide synthases are regulated to some degree, either directly or indirectly, by the presence of calcium. Also, Schuman et. al., Annual Reviews of Neuroscience, 17, 153-83, 1994 showed that phosphorylation of Type I NOS may form an additional mechanism for regulating its activity. Methods for extraction and purification of the three types of NOS are given by Fosterman et. al., Methods in Enzymology, 233:258-64, 1994. It should also be noted that NOS is regulated by the presence of NO according to Rengasamy et. al., Molecular Pharmacology, 1993, Jul., 44 (1): 124-8.
The current technology for the delivery of nitric oxide for medical applications generally relies on polymers for implant applications and does not anticipate the need for the presence of calcium and/or phosphate to regulate nitric oxide or nitric oxide precursors. Likewise, for non-implant applications nitric oxide components are generally delivered by inhalation, or as free pharmaceutical agents. Also, nitric oxide or substances containing nitric oxides, have not been previously encapsulated in or used in conjunction with ceramic or glass materials for the delivery of nitric oxide for medical applications.
Accordingly, a need exists for new implantable substrates as well as methods of effectively delivering NO to a desired site. The present invention satisfies this need and provides related advantages as well.